Enzymes are widely used as labels in immunoassay and in nucleic acid testing as labels. The catalytic activity of an enzyme can provide inherent signal amplification since one enzyme molecule can turn over many substrate molecules to produce a detectable product. The two most commonly used labels are alkaline phosphatase and horseradish peroxidase.
The enzymes alkaline phosphatase and horseradish peroxidase have been used as labels in immunohistochemistry and in immunoassays for over 30 years. Various methods are available to detect and measure the activity of alkaline phosphatase and horseradish peroxidase based on the generation of soluble and insoluble colored products, fluorescent products, and chemi-and bioluminescent products (Kricka, Ligand-binder assays, New York: Dekker, 1985; Schuurs and van Weemen, J Immunoassay 1980;1:229-49).
Chemiluminescent assays based on adamantyl 1,2-dioxetane aryl phosphates are one of the principal types of assays for detecting and measuring the activity of alkaline phosphatase. They are widely used in high throughput clinical immunoassay analyzers and commercial Western blotting kits for proteins and Southern and Northern blotting kits for nucleic acids. Alkaline phosphatase cleaves the phosphate group to produce an unstable phenoxide. This decomposes via scission of the 1,2-dioxetane ring to produce adamantanone and a fluorescent phenoxy ester in an electronically excited state. The latter intermediate decays to the electronic ground state and the excess energy is emitted as a glow of light (Bronstein et al. J Biolumin Chemilumin 1989;4:99-111). It should be appreciated that the chemiluminescent 1,2-dioxetane type of reaction used to measure alkaline phosphatase is less than ten percent efficient. Ninety percent of the substrate that reacts with the enzyme does not produce a light signal. Any means of improving the light producing efficiency of this reaction would be rewarded in improvements in the detection limit for alkaline phosphatase and this would translate into improved sensitivity for assays using this label.
Chemiluminescent assays based on the luminol reaction enhanced by phenols are one of the principal types of assays for detecting and measuring the activity of horseradish peroxidase and are widely used in high throughput routine clinical immunoassay analyzers and commercial Western blotting kits for proteins and Southern and Northern blotting kits for nucleic acids.
The chemiluminescent luminol reaction used to measure peroxidase is less than one percent efficient. Ninety-nine percent of the substrate that reacts with the enzyme does not produce a light signal. Any means of improving the light producing efficiency of this reaction would be rewarded in improvements in the detection limit for horseradish peroxidase and this would translate into improved sensitivity for assays using this label.
The gene for firefly luciferase has become important as a reporter gene (Bronstein et al., Biotechniques 1994;17:172-4, 176-7), and gene expression is assessed by measuring the activity of the expressed firefly luciferase using a mixture of Mg-ATP and firefly luciferin. The reaction of firefly luciferase with firefly luciferin is the most efficient bioluminescent reaction known and the quantum yield has been estimated to approach 100%. A feature of this bioluminescent reaction is that the product, oxyluciferin, is an inhibitor of the enzyme, and thus it only turns over a few times before becoming inactivated. This, together with the difficulty encountered in attaching antigen and antibodies to the very hydrophobic luciferase, has limited the use of this enzyme as a label. A means for improving the turn-over of this enzyme would be rewarded in improvements in the detection limit for luciferase.
As will be appreciated, there are many other compounds that can be used to generate luminescence in luminescent assay systems. The full potential of these compounds remains unrealized. A need exists for improving the efficiency of luminescent assays. The present invention is directed to this, as well as other, important ends.